The long-term objective of the proposed research is to elucidate the mechanisms of xenobiotic- mediated inactivation, degradation, and turnover of cytochrome P450 enzymes. Nitric oxide synthase (NOS), the most highly regulated cytochrome P450 enzyme, plays a key role in a variety of biological processes, including regulation of gastrointestinal motility and liver drug metabolism. We have discovered that drugs, such as guanabenz and tobacco, are metabolism- based inactivators of neuronal NOS (nNOS) and lead to the covalent alteration, enhanced turnover, and loss of nNOS P450 protein via the ubiquitin proteasomal pathway. The loss of NOS is a mechanism of toxicity associated with these drugs. We have established that alteration of the active site conformation 'labilizes' the nNOS, which is then recognized by Hsp70 and Hsp90 chaperones, and is ubiquitinated by CHIP, a chaperone-associated ubiquitin ligase, resulting in the specific proteasomal degradation of the labilized nNOS. We plan on utilizing these discoveries and our recent ground-breaking success with electron microscopy (EM) studies on nNOS and nNOS?Hsp70?CHIP complexes to better understand how chaperones recognize labilized nNOS P450 through the following specific aims: (1) To characterize the structures of the stabilized and labilized states of nNOS with the use of single particle negative stain EM and cryogenic-EM techniques, (2) To characterize the structure of nNOS chaperone complexes with Hsp70 and Hsp90 by EM as well as LC-MS/MS techniques, (3) To isolate and characterize the chaperones, co-chaperones and other proteins that associate with labilized nNOS by use of a cell permeable thiol-cleavable crosslinker and LC- MS/MS methods. This work would be the first to elucidate the structure of full-length nNOS, nNOS?chaperone complexes, as well as determine the specific conformational states of nNOS that are recognized by chaperones. These studies should lead to a better understanding of how chaperones recognize labilized forms of nNOS and maintain protein quality. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. Moreover, understanding the mechanism of recognition of labilized nNOS and quality control may provide a new method to specifically remove proteins for therapeutic benefit. An example of such utility is our recent study on removal of protein aggregates through activation of chaperones in a neurodegenerative disease model (Nature Chemical Biology 9: 112-118, 2013).